Gene silencing by siRNA (short interfering RNA) is a developing field in biology and has evolved as a novel post-transcriptional gene silencing strategy with therapeutic potential. Based on the sequencing of the human genome and the understanding of the molecular causes of diseases, the possibility of turning off pathogenic genes at will is an appealing approach for treatment of a wide variety of clinical pathologies, such as diabetes, atherosclerosis and cancer. With siRNAs, virtually every gene in the human genome contributing to a disease becomes amenable to regulation, thus opening opportunities for drug discovery. Whereas locally administered siRNAs have already entered the first clinical trials, strategies for successful systemic delivery of siRNA are still in a preclinical stage of development.
Type II Diabetes Mellitus
Type II diabetes mellitus (T2DM) is a progressive metabolic disorder with diverse pathologic manifestations and is often associated with lipid metabolism and glycometabolic disorders (Bell et al., 2001, Nature, 414:788-791). Type II diabetes is characterized by a resistance to insulin action in peripheral tissues such as muscle, adipose tissue and liver. It is also characterized by a progressive failure in the ability of the islet β-cell to secrete insulin. The long term effects of diabetes result from its vascular complications; micro vascular complications, retinopathy, neuropathy and nephropathy. Macro vascular complications are associated with type II diabetes as well, and include cardiovascular and cerebrovascular complications.
The main classes of anti-diabetic drugs known today are the following. Biguanides are a class of drugs that help control blood glucose by inhibiting hepatic glucose production, reducing intestinal absorption and enhancing peripheral glucose uptake. This class includes metformin, a drug that lowers both glucose and blood triglycerides level. Sulfonylurea is a class of drugs that helps in controlling or managing type II diabetes by stimulating the release of endogenous insulin from the β-cells of the pancreas. This class includes: tolbutamide, tolazamide, glisoxepide, glimipeide and glibomuride among others. Glycosidase inhibitors stimulate the release of insulin from pancreatic cells thus lowering blood sugar level and include repaglinide and nateglinide.
Unfortunately, these treatment modalities, even when combined, are frequently constrained by safety, tolerability, weight gain, oedema and gastrointestinal intolerance (Drucker et al., 2010, Nat Rev Drug Discov, 9:267-268; Nauck et al., 2009, Diabetes Care, 32:84-90; Ng et al., 2010, Prim Care Diabetes, 4:61-63; Truitt et al., 2010, Curr Med Res Opin, 26:1321-1331; and Wajcberg and Tavaria, 2009, Expert Opin Pharmacother, 10:135-142). In addition, as the disease progresses and β-cell function declines, efficacies of current treatments diminish (Turner et al., 1999, JAMA, 281:2005-2012).
The discovery of the incretin effect has provided a new avenue of treatment using a class of therapeutics capable of controlling T2DM with minimal adverse effects. The incretin effect is mainly mediated by glucagon like peptide 1 (GLP-1) which regulates postprandial blood glucose level via the stimulation of insulin secretion. GLP-1 has also indirect effects such as delay of gastric emptying, promoting satiety through its effect on the central nervous system, promoting β-cell growth and inhibiting β-cell apoptosis as demonstrated in animal models (Nauck et al., 2002, J Clin Endocrinol Metab, 87:1239-1246; and Creutzfeldt et al., 1996, Diabetes Care, 19:580-586). However, the potential of GLP-1 in the clinic was hindered due to its rapid degradation by the ubiquitous serine protease dipeptidyl peptidase IV (DPP-IV). The discovery that DPP-IV cleaves the His:Ala:Glu sequence at the N-terminal region of GLP-1 permitted the development of DPP-IV resistant GLP-1 analogues and the development of DPP-IV inhibitors.
DPP-IV inhibitors are a new class of drugs that inhibit the proteolytic activity of dipeptidyl peptidase IV. The proteolytic activity of DPP-IV decreases blood level of glucoregulatory peptides, known as incretins. Inhibition of dipeptidyl peptidase IV thereby potentiates the action of these incretin, notably glucagon like peptide 1 (GLP-1). These inhibitors include Sitagliptin, Vildagliptin and Saxagliptin and are orally administrated once daily.
Atherosclerosis
Atherosclerosis is a chronic disease caused by the formation of atherosclerotic plaque in arteries. Atherosclerosis represents a multitude of cardiovascular diseases such as coronary heart disease, acute coronary syndrome and angina pectoris (Lloyd-Jones et al., 2010, Circulation, 121:e46-e215). In the United-States, the predicted economic cost of atherosclerosis for 2010 was US$503 billion, mainly due to direct medical and indirect productivity costs (Lloyd-Jones et al., 2010, Circulation, 121:948-954). Although causal factors for atherosclerosis remain unknown, increasing evidence suggest a high role of dyslipidemia, hyperlipidemia and inflammation in the pathogenesis of this disease (Hanson et al., 2006, Nat Rev Immunol, 6:508-519; Montecucco and Mach, 2008, Clin Intery Aging, 3:341-349). Currently, the reduction of morbidity and mortality due to atherosclerosis and related pathologies—Cardiovascular Diseases (CVD)—are mainly attributable to the aggressive clinical use of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reducatase inhibitors commonly named statin-based therapies (Vermissen et al., 2008, BMJ, 337:a2423). These therapies reduce low density lipoprotein cholesterol (LDL-C). Intervention studies have demonstrated reduced risk of CVD morbidity and mortality when lipid lowering therapies were administered. Additionally, the decreased morbidity/mortality and LDL-C lowering demonstrate a log-linear association (Law et al., 1994, BMJ, 308:367-372).
An alternative approach to lowering LDL-C, and thus reducing atherosclerosis, is the inhibition or blocking of very low density lipoprotein (VLDL) secretion from the liver. This inhibition can be achieved through apolipoprotein B (ApoB) targeting since ApoB is necessary for VLDL secretion (Rutledge et al., 2010, Cell Biol, 88:251-267). ApoB is mainly expressed by hepatocytes and entherocytes in humans.
In humans, the ApoB gene is located on chromosome 2 (2q) and spans over 43 kb. ApoB mRNA consists of 28 introns and 29 exons and is characterized by a 16 hour half life (Ludwig et al., 1987, DNA, 6:363-372; Scott, 1989, Curr Opin Cell Biol, 1:1141-1147). The translation of ApoB mRNA yields a protein with 4,536 amino acids and an apparent molecular weight of 517-550 kDa thus representing one of the largest monomeric proteins. The importance of ApoB inhibition as an alternative therapy for atherosclerosis and its associated CVDs resides in the ability of ApoB to physically interact through its β-sheet domains with lipids such as phospholipids, cholesterol and cholesteryl esters to form large lipoproteins particles, namely VLDL, in the liver and cholymicrons in the intestine (reviewed in Rutledge et al., 2010, Biochem Cell Biol, 88:251-267).
Cancer
Classical cancer therapy includes the use of one or several chemotherapeutic drugs. These treatment modalities are associated with toxicity and severe side effects due to their non-specificity. Another major problem associated with chemotherapy is the development of chemoresistance with time. For example, resistance to chemotherapy is one of the major problems associated with the management of breast cancer.
Cancer cells employ a plethora of mechanisms to acquire resistance to one or more chemotherapeutic agent. Major mechanisms of drug resistance include (1) decreased intracellular uptake of soluble drugs, (2) genetic and phenotypic changes in cells that change the capacity of drugs to cause the desired cell damage and (3) increased efflux of drugs by cell-surface transporters, leading to multidrug resistance (MDR). In all these cases resistance to a single chemotherapeutic entity is always associated with a wide-range drug resistance pattern against other chemotherapeutics.
One of the most common and studied resistance mechanisms is the reduction of intracellular drug concentration by transporter proteins that pump drugs out of cells before they reach the site of action, so that the cells adapt to low drug concentration without undergoing drug-induced cell death. Most of these transporters are in the ATP-binding cassette transmembrane protein super-family.
In humans, 48 ABC genes (genes in the ATP-binding cassette family) have been identified to date. In breast cancer, practically all MDR resistance reported to date were closely related to one of the following: p-glycoprotein (P-gp), multidrug resistance-related protein (MRP), and breast cancer resistance protein (BCRP).
The P-gp is the most common protein involved in ATP-dependent efflux of drugs in various cancer tissues. The over expression P-gp was believed for some time to be the only protein capable of conferring MDR in mammalian tumor cells. In breast cancer, 52% of chemotherapy-treated patients had their P-gp up regulated due to therapy. The gene encoding P-gp is termed ABCB1 (mdr1) and is located on chromosome 7 at the position q21.12. ABCB1 is composed of 28 exons whose product yield a 1.2 kb mRNA. Protein sequence analysis of P-gp revealed the presence of two extracytoplasmic domains, each containing 6 putative transmembrane segments, and an ATP-binding consensus motif.
Furthermore, one class of interesting enzymes involved in maintenance of genomic integrity and stability are DNA helicases. These proteins play important roles in DNA replication, repair, recombination and transcription by an ATP dependant mechanism that unwinds duplex genomic strands allowing the repair machinery access to damaged or mispaired DNA.
For example, the RecQ family of helicases has been shown to play an important role in recombination, repair and Holliday junction formation. More recently, these helicases have been implicated in the process of posttranscriptional gene silencing (Cogoni and Macino, 1999, Science, 286:2342-2344). In this process, the helicase is required to separate the double stranded DNA before any hybridization and silencing mechanism could be initiated. Other roles have been put forward for proteins of this family. For example, RecQL1 is believed to play a role in nuclear protein transport since it interacts with both QIP1 and QIP2 proteins which function as nuclear localization signals as demonstrated in a two hybrid screening (Seki et al., 1997, 234:48-53).
The RecQ family consists of five members and can be divided into two groups according to whether they contain an additional carboxy- or amino-terminus group. Mutations in these genes lead to increased incidence of cancer as well as other physiologic abnormalities (Karow et al., 2000, Curr Opin Genet Dev, 10:32-38; Kawabe et al., 2000, Oncogene, 19:4767-4772). Such abnormalities include Blooms syndrome (BLM), Wemer's syndrome (WRN) and the Rothmund-Thompson syndrome (RecQ4). The human RecQL1 gene was the first human member of this family to be identified and was shown to have extensive homology with the E.coli DNA helicase, RecQ, and is located on chromosome 12p11 (Puranam and Blackshear, 1994, J Biol Chem, 269:29838-29845; Puranam et al., 1995, Genomics, 26:595-598).
RecQL1 over expression in cancerous cell lines such as AsPC1, A549 and LS174T among others is believed to be driven in order to compensate the high recombination rate in these cancerous cells, thus preventing apoptosis (Futami et al., 2008, Cancer Sci, 99:71-80). RecQL1 gene silencing using specific siRNA in these cell lines or in a murine Xenograft model lead to an increased cancerous cell death and tumor mass reduction (Futami et al., 2008, Cancer Sci, 99:71-80).
Another class of enzymes involved in maintenance of homeostatic stability and functional integrity are RNA helicases. These enzymes are characterized by the presence of a centrally located “helicase domain”, consisting of eight conserved motifs. Based on these motifs, RNA helicases are classified into families. These conserved motifs are required to perform the NTP hydrolysis and RNA unwinding functions (Linder et al., 2001, Trends Biochem Sci., 26:339-341; Tanner and Linder, 2001, Mol Cell, 8:251-262). Another function that has been associated with RNA helicases is disruption of RNA-protein interactions (Jankowsky et al., 2001, Science, 291:121-125). These enzymes are members of molecular complexes that can regulate both their NTPase and helicase activities (Silverman et al., 2003, Gene, 312:1-16). The intrinsic characteristics of these helicases play an important role in post transcriptional events since the modulation of RNA secondary structure regulates steps such as splicing (Balvay et al., 1993, Bioessays, 15:165-169) and translation (van der Velden and Thomas, 1999, Int J Biochem Cell Biol, 31:87-106).
Dysregulation of RNA processing molecules such as RNA helicase have been implicated in human pathologies and cancer development. Examples of these helicases implicated in human pathologies include DDX1/5/6/9/10 and DHX32 among others (Abdelhaleem, 2004, Anticancer Res, 2004, 24:3951-3953; Abdelhaleem, 2004, Biocim Biophys Acta, 1704:37-46). These helicases contain a characteristic DEAD box domain and are up-regulated in most cancers (Abdelhaleem, 2004, Anticancer Res, 2004, 24:3951-3953; Abdelhaleem, 2004, Biocim Biophys Acta, 1704:37-46).
There is still a need today to be provided with alternative therapies by sustaining siRNA delivery in vivo. Particularly, it would be highly desirable to be provided with an alternative means for treating type II diabetes mellitus, atherosclerosis and cancer.